Expression of Mouse IAN Family Genes

<div><p>(A) Quantitative RT-PCR analysis of total RNA from C57BL/6 mouse tissues, purified splenocyte subsets, and purified thymocyte subsets. The mRNA levels of IAN family genes were initially normalized to <i>GAPDH</i> levels, and were further normalized to the levels expressed in the thymus. Relative expression of all IAN family genes in the thymus tissue is indicated as 1. </p> <p>(B) Relative mRNA levels of <i>IAN1, IAN4,</i> and <i>IAN5</i> in CD4⁺CD8⁺, CD4⁺CD8⁺CD5^low, and CD4⁺CD8⁺CD5^high thymocytes from C57BL/6 (wild-type) mice and CD4⁺CD8⁺ thymocytes from TCRα-deficient mice [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b042" target="_blank">42</a>]. </p> <p>(C) <i>IAN1, IAN4,</i> and <i>IAN5</i> mRNA levels in CD4⁺CD8⁺ thymocytes from positive selector (AND-TCR Aβ^+/+ and 2C-TCR^k/b [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b043" target="_blank">43</a>]) TCR-transgenic mice and null selector (AND-TCR Aβ^−/− [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b044" target="_blank">44</a>] and 2C-TCR^k/k) TCR-transgenic mice. </p> <p>(D) Relative mRNA levels of <i>IAN1, IAN4,</i> and <i>IAN5</i> in total, CD4⁺CD8⁺CD69^low, CD4⁺CD8⁺CD69^high, CD4⁺CD8⁻CD69^high and CD4⁺CD8⁻CD69^low thymocytes from C57BL/6 mice. </p> <p>(E) Thymocytes from TCRα-deficient mice were cultured with or without phorbol 12-myristate 13-acetate (0.2 ng/ml) and ionomycin (0.2 μg/ml) for the indicated periods.</p> <p>(F) Thymocytes from Aβ^−/− β2m^−/− mice [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040103#pbio-0040103-b045" target="_blank">45</a>] were cultured with or without plate-bound anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) antibodies for 24 h. </p> <p>Bar graphs show means ± standard errors.</p>